Endocytosis is a fundamental process in eukaryotic cells important for a variety of cellular functions ranging from the acquisition of certain nutrients, regulation of cell function, including cholesterol synthesis related to atherosclerotic plaque formation in heart disease, and response of the immune system in antibody production. The overall objective of this proposal is to better understand basic molecular events underlying steps of the endocytic process. Our approach is to isolate temperature-sensitive mutants of mammalian cells that are defective in some aspect of endocytosis and then study the mutant cells to gain information about the specific events that are defective. In specific aim one, we propose a variety of physiological and morphological studies in vivo to characterize a mutant, termed V.24.1, that has defective lysosomes at the defect. In addition, we plan an in vitro approach to investigating protein targeting in the mutant cells. Specific aim two involves studying in vitro complementation between two other mutants that are defective in endosomal acidification. In specific aim three we propose to further characterize another mutant, termed 42.2, that has an undefined lesion affecting endocytosis. Specific aim four involves using a new technique we have devised to isolate mutants expressing temperature-sensitive lesions affecting early steps of endocytic vesicle formation.